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Salmonella Blockley is rarely isolated in the Western Hemisphere. According to Enter-net, the international network for surveillance of Salmonella and verocytotoxin-producing Escheri-chia coli infections, S. Blockley represented 0.6% of all Salmonella serotypes isolated in Europe during the first quarter of 1998, a full 100-fold lower than the dominant serotype, S. Enteritidis (67.1%) (1). However, S. Blockley is among the five most frequently isolated serotypes from both avian and human sources in Japan (2,3), Malaysia (4,5), and Thailand (6). A single foodborne outbreak in the United States (7) and sporadic human infections in Europe associated with travel to the Far East (8), animal infection (9) or carriage (10,11), and environmental isolates have also been reported (12,13). Regardless of the frequency of S. Blockley isolation, its rates of resistance to antibiotics have been high. Among Spanish salmonellae isolated from natural water reservoirs, S. Blockley and S. Typhimurium had the highest rates of multidrug resistance (12). Comparing 1980-1989 with 1990-1994, researchers from Tokyo noted an increase in the number of S. Blockley isolates resistant to one or more antibiotics, from 92.0% to 98.2% for imported cases and from 57.4% to 88.7% for domestic cases (3,14). In Thailand, isolates from human or other sources also had high rates of resistance to streptomycin, tetracycline, kanamycin, and chloramphenicol and lower rates to ampicillin and trimethoprim/sulfamethoxazole (15). Nevertheless, few attempts at typing S. Blockley isolates with molecular methods have been described, and these have been limited to the characterization of plasmid content (2,16). During the second and third quarters of 1998, Enter-net reported higher numbers of S. Blockley isolates than during the same period of the previous year in several European countries (1). The epidemiologic investigations conducted in Germany, England and Wales, and Greece did not confirm a source for this increase (17-19). In this study, we characterized the Greek outbreak isolates further, both with respect to their antibiotic resistance phenotypes and DNA fingerprints obtained by pulsed-field gel electro-phoresis of genomic DNA. The Study The study sample consisted of 28 of 35 S. During 1998, a marked increase (35 cases) in human gastroenteritis due to Salmonella Blockley, a serotype rarely isolated from humans in the Western Hemisphere, was noted in Greece. The two dominant multidrug-resistance phenotypes (23 of the 29 isolates studied) were associated with two distinct DNA fingerprints, obtained by pulsed-field gel electrophoresis of genomic DNA.


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were assigned to isolates with electrophoretic patterns differing by one to three DNA fragments (23). Gel images were also processed by the GelCompar software (Applied Maths, Kortrijk, Belgium), and on the basis of PFGE pattern similarities, a dendrogram was constructed by using the Dice coefficient and clustering by the unweighted pair group method, which uses arithmetic averages (UPGMA) with a 2% tolerance in band position difference.
control strains: one from 1996 and three from 1997. All isolates were from human cases of enteritis. Identification was performed by the API 20E system (BioMerieux S.A., Marcy l'Etoile, France) and serotyping with commercially obtained antisera (BioMerieux) (20). Susceptibility to kanamycin, streptomycin, ampicillin, amoxicillin/clavulanic acid, cefepime, tetracycline, chloramphenicol, trimethoprim/ sulfamethoxazole, gentamicin, nalidixic acid, and ciprofloxacin was tested by a disk diffusion assay according to National Committee for Clinical Laboratory Standards guidelines (21). Genomic DNA was prepared and digested with XbaI (New England Biolabs) (22). Chi-square tests or Fisher exact tests were used to calculate two-tailed probabilities.
S. Blockley accounted for seven of the 13,199 salmonella isolates identified in Greece from 1976 to 1997. However, 35 gastroenteritis cases due to this serotype were reported from May to December 1998 (19). Twenty-nine S. Blockley strains isolated from fecal specimens of patients with gastroenteritis during May 1998 to February 1999, along with four epidemiologically unrelated clinical isolates from 1996 and 1997, were therefore studied for susceptibility to antibiotics. The 1998 outbreak isolates were scattered throughout Greece; S. Blockley was isolated later, starting in August 1998, in northern Greece.
When pulsed-field gel electrophoresis was used to obtain DNA fingerprints for these isolates (Figure 1), all belonged to the same type, A, although eight subtypes, A1-A8, could be distinguished on the basis of one to three DNA fragment differences (Figures 1, 2). Two of the four isolates from previous years belonged to unique subtypes A6 and A7; the other two belonged to subtypes A2 and A8, shared by outbreak isolates (Table). In contrast, 93% of the 1998 outbreak strains yielded PFGE patterns common to two or more isolates. Indeed, most outbreak isolates were grouped in subtypes A2 and A4, consisting of 11 and 12 isolates, respectively (Table).

Conclusions
Our results indicate that PFGE is useful in distinguishing epidemiologically related S. Blockley isolates since two of the four nonoutbreak isolates displayed unique PFGE patterns, A7 and A8, while PFGE patterns A2 and A4 grouped most of the 29 outbreak isolates (11 and 12, respectively).
These two chromosomal fingerprints, differing by two DNA fragments, were associated with two distinct resistance phenotypes. The resistance phenotype of A4 isolates, ATCN, was identical to the earlier resistance phenotype of A2 isolates, ATC, except for the resistance to nalidixic acid. Nevertheless, these two PFGE/ antibiotic resistance types, A2/ATC and A4/ ATCN, displayed a clear distribution both in time and space.
The data may, therefore, indicate two main sources for the outbreak. Alternatively, and perhaps more likely, these two closely related types may together constitute the outbreak clone, evolved with time to acquire resistance to nalidixic acid. Resistance may well have originated in the food source, since several antibiotic classes are used as feed supplements in animal rearing and aquaculture in Greece: sulfonamides (trimethoprim/sulfathiazine), tetracyclines (oxytetracycline), and quinolones (oxolinic acid). However, as in other European countries (17,18), the epidemiologic investigation did not locate a common source to account for the wide geographic spread of cases (19). Although travel was not mentioned in the Greek patients' questionnaire responses, the possibility that the source was an imported food cannot be ruled out. The association with smoked eel of Italian origin in the German outbreak has not been microbiologically confirmed (17). The only other previous European report of a human outbreak attributed to S. Blockley, probably from vegetables contaminated by this organism, which was prevalent in irrigation water in the Spanish region of Granada, is anecdotal (13). A documented S. Blockley enteritis epidemic in a U.S. hospital in 1966 was attributed to contaminated ice cream; however, this was also not microbiologically confirmed (7).
While this serotype may remain important in Europe, its high rates of resistance to kanamycin, streptomycin, tetracycline, and chloramphenicol, which were in agreement with studies from the Far East (3) and Spain (12), are cause for concern. Unlike the Far Eastern strains, no resistance to ß-lactam antibiotics or cotrimoxazole was observed in our study. The

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two dominant resistant phenotypes of S. Blockley from natural polluted waters in Spain were sulfonamides, streptomycin, and tetracycline; and neomycin, streptomycin, kanamycin, tetracycline, and chloramphenicol (12), as in the Greek strains, except for the absence of resistance to nalidixic acid.
In agreement with differences in animal reservoirs and transmission routes and therefore the mechanism of resistance acquisition among different Salmonella serotypes, the main patterns of resistance observed in S. Blockley were distinct from those predominating in the two major serotypes from isolates of both human and animal food origin in Greece. In S. Enteritidis, the most frequent resistance phenotype was resistance to ampicillin (24), while in S. Typhimurium, the most frequent resistant phenotype was resistance to sulfonamides and streptomycin (A. Markogiannakis, P.T. Tassios, N.J. Legakis, unpub. obs.). Furthermore, the considerably high rate of resistance to nalidixic acid is equally unprecedented in both the Far Eastern and Spanish S. Blockley isolates and in other salmonella serotypes from Greece. Since resistance to nalidixic acid can be a precursor of resistance to fluoroquinolones, one of the two drug classes of choice for invasive salmonella disease, this feature of these S. Blockley strains is particularly disturbing. S. Blockley, previously a prevalent serotype in the Far East but rare elsewhere, nevertheless posed a public health problem in several European countries. The source of the European outbreaks, however, remains unclear. Given the increased international commerce in food, a collaborative study would be useful in identifying potential similarities between the recent European strains and established strains from the Far East.